Porous and bulk keratin bio-polymers

ABSTRACT

Methods for producing thin keratin films, sheets, and bulk materials, and products formed using these methods. One method includes providing hair, reducing the hair such that the disulfide linkages are broken and free cysteine thiol groups formed, separating out a more soluble keratin fraction in solution, forming a thin layer from the more soluble fraction, and air drying the keratin fraction in the presence of oxygen, thereby forming new disulfide bonds imparting strength to the resulting thin keratin film. One method includes reducing hair by heating the hair under nitrogen in an ammonium hydroxide and ammonium thioglycolate solution followed by centrifuging and collecting the supernatant containing the more soluble keratin fraction. The more soluble keratin in this method is precipitated using HCl, removed, and resuspended in ammonium hydroxide. The keratin solution thus formed is poured onto a flat surface and allowed to air dry into a thin keratin film. The film may be used as a wound dressing, a tissue-engineering scaffold, a diffusion membrane, a coating for implantable devices, and as a cell encapsulant. In another method, the keratin solution thus formed is concentrated, poured into a mold, and allowed to air dry into a three dimensional keratin product. The resulting bulk product can be used as a cross-linked implantable biomaterial for soft and hard tissue replacement. In another method, a keratin solution is emulsified and freeze dried, forming a porous, open cell keratin material.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/198,998, filed Nov. 24, 1998, now U.S. Pat. No. 6,110,487 which inturn is a continuation-in-part of U.S. Pat. No. 5,948,432, which wasfiled as U.S. patent application Ser. No. 08/979,526, on Nov. 26, 1997.The present application is related to U.S. patent application Ser. No.09/057,161, filed Apr. 8, 1998, entitled KERATINOUS PROTEIN MATERIAL FORWOUND HEALING APPLICATIONS; U.S. patent application Ser. No. 08/979,456,filed Nov. 26, 1997, entitled KERATIN-BASED HYDROGEL FOR BIOMEDICALAPPLICATIONS AND METHOD OF PRODUCTION; and U.S. Pat. No. 5,358,935,entitled NONANTIGENIC KERATINOUS PROTEIN MATERIAL, all hereinincorporated by reference.

FIELD OF THE INVENTION

The present invention is related to products formed from Keratin derivedfrom hair. More specifically, the present invention is related to films,sheets, and bulk materials formed from keratin. The present invention isdirected to a cross-linked keratin based bulk, film or sheet materialfor use in biomedical implant, wound dressing, and tissue engineeringapplications. More specifically, one aspect of the present inventionrelates to a material based primarily on alpha keratin produced bycross-linking keratin derived from a soluble fraction of keratinousmaterial such as hair.

BACKGROUND OF THE INVENTION

Chronic wounds can be caused by a variety of events, including surgery,prolonged bed rest, and traumatic injuries. Partial thickness wounds caninclude second degree burns, abrasions, and skin graft donor sites.Healing of these wounds can be problematic, especially in cases ofdiabetes mellitus or chronic immune disorders. Full thickness woundshave no skin remaining, and can be the result of trauma, diabetes (e.g.,leg ulcers), and venous stasis disease, which can cause full thicknessulcers of the lower extremities. Full thickness wounds tend to heal veryslowly. Proper wound care technique, including the use of wounddressings, is extremely important to successful chronic woundmanagement. Chronic wounds affect an estimated four million people ayear, resulting in health care costs in the billions of dollars."Treatment of Skin Ulcers with Cultivated Epidermal Allografts," T.Phillips, O. Kehinde, and H. Green, J. Am. Acad. Dermatol., V. 21, pp.191-199 (1989).

The wound healing process involves a complex series of biologicalinteractions at the cellular level, which can be grouped into threephases: hemostasis and inflammation, granulation tissue formation andreepithelization; and remodeling. "Cutaneous Tissue Repair: BasicBiological Considerations," R. A. F. Clark, J. Am. Acad. Dermatol., Vol.13, pp. 701-725 (1985). Keratinocytes (epidermal cells that manufactureand contain keratin) migrate from wound edges to cover the wound. Growthfactors such as transforming growth factor-β (TGF-β) play a criticalrole in stimulating the migration process. The migration occursoptimally under the cover of a moist layer. Keratins have been found tobe necessary for reepithelization. Specifically, keratin types K5 andK14 have been found in the lower, generating, epidermal cells, and typesK1 and K10 have been found in the upper, differentiated cells. WoundHealing: Biochemical and Clinical Aspects, I. K. Cohen, R. F. Diegleman,and W. J. Lindblad, eds., W. W. Saunders Company, 1992. Keratin types K6and K10 are believed to be present in healing wounds, but not in normalskin. Keratins are major structural proteins of all epithelial celltypes and appear to play a major role in wound healing.

An optimum wound dressing would protect the injured tissue, maintain amoist environment, be water permeable, maintain microbial control,deliver healing agents to the wound site, be easy to apply, not requirefrequent changes, and be non-toxic and non-antigenic. Although not idealfor chronic wounds, several wound dressings are currently on the market,including occlusive dressings, non-adherent dressings, absorbentdressings, and dressings in the form of sheets, foams, powders, andgels. Wound Management and Dressing, S. Thomas, The PharmaceuticalPress, London, 1990.

Attempts have been made to provide improved dressings that would assistin the wound healing process using biological materials such as growthfactors. To date, these biologicals have proven very costly and haveshown minimal clinical relevance in accelerating the chronic woundhealing process. In cases of severe full thickness wounds, autografts(skin grafts from the patient's body) are often used. Although the graftis non-antigenic, it must be harvested from a donor site on thepatient's body, creating an additional wound. In addition, availabilityof autologous tissue may not be adequate. Allografts (skin grafts fromdonors other than the patient) are also used when donor sites are not anoption. Allografts essentially provide a "wound dressing" that providesa moist, water-permeable layer, but is rejected by the patient usuallywithin two weeks and does not become part of the new epidermis.

What would be desirable, and has not heretofore been provided, is awound dressing that protects the injured tissue, maintains a moistenvironment, is water permeable, is easy to apply, does not requirefrequent changes, is non-toxic and non-antigenic, and most important,delivers effective healing agents to the wound site.

Film materials compatible with living tissue are useful for a number ofapplications including tissue engineering scaffolding, diffusionmembranes, coatings for implantable devices, and cell encapsulants. Bulkkeratin materials compatible with living tissue are useful for a numberof applications including open cell tissue engineering scaffolding andbulk, cross-linked biomaterials. Tissue engineering is a rapidly growingfield encompassing a number of technologies aimed at replacing orrestoring tissue and organ function. The consistent success of atissue-engineered implant rests on the invention of a biocompatible,mitogenic material that can successfully support cell growth anddifferentiation and integrate into existing tissue. Such a scaffoldingmaterial could greatly advance the state of the tissue engineeringtechnologies and result in a wide array of tissue engineered implantscontaining cellular components, such as osteoblasts, chondrocytes,keratinocytes, and hepatocytes, to restore or replace bone, cartilage,skin, and liver tissue respectively.

Diffusion membranes are commonly formed of synthetic polymericmaterials, rather than biologically-derived materials. Diffusionmembranes derived from biological materials have the advantage ofenhanced biocompatibility. In particular, non-antigenic diffusionmembranes are compatible with implantation in the human body and wouldprovide great advantages in controlled drug release applications.

Implantable devices, such as pacemakers, stents, orthopedic implants,urological implants, dental implants, breast implants, and implants formaxillofacial reconstruction are currently encased in, or made of,materials including titanium, silicone, stainless steel, hydroxyapatite,and polyethylene, or encapsulated in materials such as silicone orpolyurethane. These metals, ceramics, and synthetic polymers havedisadvantages related to biocompatibility and antigenicity which canlead to problems related to the long term use of these devices. Acoating material derived from biological materials and havingnon-antigenic and mitogenic properties would provide a device theadvantage of long term biocompatibility in vivo and potentially extendthe useful lifetime of an implant while decreasing the risk of anallergic or negative immune response from the host.

Cell encapsulants such as Chitin/Alginate and bovine-derived collagenare used to encapsulate mammalian cells for applications such as tissueengineering/organ regeneration and bacteria for cloning applications. Anon-antigenic, non bioresorbable cell encapsulant material would havethe advantages of providing the cell with a mitogen and increasing thechances for the cell to accomplish its tissue engineering function.

A bulk, cross-linked implantable biomaterial that was non-antigenic andpossessed the appropriate mechanical properties could be used formaxillofacial restoration, for example, for both soft and hard tissuereplacement. Such a bulk material could also be used for orthopedicapplications as a bone filler and for cartilage regeneration. A bulkmaterial capable of being implanted could also be used for neurologicalapplications, such as for nerve regeneration guides.

Keratin, often derived from vertebrate hair, has been processed intovarious forms. Commonly assigned U.S. Pat. No. 5,358,935 disclosesmechanically processing human hair into a keratinous powder. The hair isbleached, rinsed, dried, chopped, homogenized, ultrasonicated, andremoved from solvent, leaving a keratin powder. In U.S. Pat. No.5,047,249, Rothman discusses activating keratin with a reducing agentand applying the activated keratin to a wound. Rothman believes theactivated keratin thiol groups will react with thiol groups in the woundtissue and form a disulfide bond, allowing the keratin to adhere to andprotect the wound.

Keratin derived materials are believed to be non-antigenic, particularlywhen derived from a patient's own keratin. A film formed from keratinbased material would be desirable. A keratin film able to be used fortissue-engineering scaffolds, diffusion membranes, implantable devicecoatings, and cell encapsulants would be very useful. A solid keratinbulk material would also have great utility. In addition, anon-antigenic, mitogenic open cell keratin scaffold would prove highlybeneficial for use as a tissue engineered scaffold to support, nourish,and stimulate cell growth preceding and following implantation.

SUMMARY OF THE INVENTION

The present invention includes a sheet formed of cross-linked keratinnot requiring a synthetic binding agent. The sheet is believed to bebound together by reformed disulfide linkages and hydrogen bonds. Apreferred use of the sheet is as a wound healing dressing. Anotherpreferred use is as a tissue engineering cell scaffold for implantapplications. The sheet can be formed from a combination of soluble andinsoluble protein fractions derived from hair, including alpha and betakeratin fractions. Keratin can be obtained from a number of sources,including human or animal hair and finger or toe nails, with one sourcebeing hair of the patient or a donor.

The sheet can be formed by providing an insoluble chemically modifiedkeratin fraction suspended in water and lowering the pH until thekeratin protein is partially swelled. Partially swelled is defined asthe protein molecule swelling such that the resulting suspension ofkeratin particles behaves like a colloidal suspension. In one method,concentrated sulfuric acid is added until a pH of less than 1 isreached. Applicants believe the low pH disrupts the hydrogen bonds whichhave been rendering the keratin fraction insoluble, thereby allowing theprotein to partially swell. The partially swelled keratin is then madebasic with ammonium hydroxide. This treatment exchanges the non-volatileacid with a volatile base, which is removed upon drying. Alternatively,a volatile acid, such as formic acid, may be employed, eliminating therequirement for further treatment with a volatile base. The resultingslurry can then be cast onto a flat surface or mold of appropriategeometry and surface finish and air dried to produce a cross-linkedkeratin sheet. Applicants believe the cross-links result from the thiolgroups re-forming disulfide linkages and from the amine, and carboxylicacid groups forming hydrogen bonds.

The resulting sheet is thus formed of pure keratin. Keratin has beenshown to be biocompatible, non-immunogenic, not to inhibit activatedT-cells and therefore not interfere with the normal cell mediated immuneresponse, and to be mitogenic for keratinocytes, fibroblasts, and humanmicrovascular endothelial cells. Keratin has also been shown to promoteepithelialization in wound healing studies on rats and humans.

Another embodiment of the invention includes partially oxidizing thekeratin disulfide linkages to form hydrophilic groups. One such methodincludes treating the keratin with peractic acid to form sulfonic acidgroups from a substantial portion, but not all of, the disulfide bonds.Most of the sulfonic acid groups remain in the final product ashydrophilic groups, binding water and hydrating the keratin material. Alater reduction step cleaves many of the remaining disulfide bonds toform cysteine residues. The partially oxidized and reduced keratin canthen be in put in solution, concentrated, and cast onto a flat surfaceto oxidize and re-form disulfide cross-links. In one method, oxygen inair acts as the oxidizing agent, with the keratin being air dried toform a film on the flat surface. The moist keratin sheet, consistingprimarily of keratin derived from beta keratin, has the consistency ofmoist, thick paper. The sheet dries to a brittle material, which can berehydrated to a supple, skin-like material. The rehydrated sheet has thelook and feel of skin while retaining moisture within the sheet andwithin the wound. The sheet can be used as a wound-healing dressing oras a cell-growth scaffold. The sheet can be cut and shaped as neededbefore being applied to the wound. The keratin sheets provide anon-antigenic wound dressing that maintains wound moisture for migratingepithelial cells and provides a scaffold for cell growth for tissueengineered implants. Other applications for this keratin sheet includeuse as diffusion membranes and as an encapsulant for cells.

The present invention includes methods for forming keratin based thinfilms, open cell foams, and bulk materials. The thin films are suitablefor use as wound dressings, tissue-engineering scaffolds, diffusionmembranes, coatings for implantable devices, and cell encapsulants. Inone method, cut, washed, rinsed, and dried vertebrate hair is provided.The hair is reduced with a reducing agent, such that some of thedisulfide linkages are broken, and a more soluble keratin fraction and aless soluble keratin fraction formed. The more soluble keratin fractionis separated, collected, and deposited onto a surface, thereby forming alayer of the more soluble keratin fraction. The keratin layer is exposedto an oxidizing agent, such as air, oxygen, or H₂ O₂, and preferablydried. The free thiol groups are oxidized by the oxidizing agent, theresulting keratin film being strengthened by the newly formed disulfidebonds. A higher degree of crosslinking, and therefore strength, can beobtained by the addition of crosslinking agents such as glutaraldehyde.

In one method according to the present invention, a keratin solution isprovided, the keratin being dissolved in a first solvent such aqueousthioglycolate. The keratin has free thiol groups, produced by methodssuch as reduction with ammonium thioglycolate. A second solvent such ashexane or Freon is provided, the second solvent preferably beingsubstantially immiscible in the first solvent and the keratin preferablybeing substantially insoluble in the second solvent. An emulsion of thesecond solvent in the keratin solution can be formed using ahomogenizer. The emulsion is freeze dried, preferably by freezing theemulsion and removing the first and second solvents under vacuum,creating a porous keratin material. The porous keratin material can bewarmed to room temperature in the presence of an oxidizing agent,promoting the formation of disulfide cross-links between the keratin. Inone method, the oxidizing agent is an oxygen containing gas such as air.In another method, hydrogen peroxide is mixed with the keratin solutionprior to homogenizing. Applicants believe the resulting material is anopen cell scaffold having substantially spherical voids corresponding tothe second solvent in the emulsion and a cross-linked keratin structurecorresponding to the keratin solution in the emulsion.

In another method according to the present invention, a keratin solutionis provided, the keratin being dissolved in a solvent such as aqueousthioglycolate. The keratin has free thiol groups, produced by methodssuch as reduction with ammonium thioglycolate. The keratin can beatomized and sprayed onto a very cold surface, sufficiently cold tofreeze the keratin solution. In one method, the surface is the surfaceof a mold. More keratin solution can be atomized and sprayed over thealready frozen keratin, thereby building up a thicker open cell layer offrozen keratin. The frozen keratin can be freeze dried by removing atleast a substantial portion of the solvent, and preferably all of thesolvent, under low pressure at low temperature. The keratin material canbe warmed to room temperature in the presence of an oxidizing agent,promoting the formation of disulfide cross-links within the keratinsolids formed and between the keratin solids formed. In one method, theoxidizing agent includes gaseous oxygen. In another method, theoxidizing agent includes hydrogen peroxide added to the keratinsolution. Applicants believe the resulting structure is an open cellscaffold having substantially spherical keratin structures correspondingto the atomized keratin and having voids therebetween. Applicantsbelieve the substantially spherical keratin structures have disulfidecross-links formed within, and the structures have disulfide cross-linksbetween structures where touching each other.

In one method, according to the invention, hair is cut, washed, dried,and suspended in ammonium hydroxide containing ammonium thioglycolate.The suspension is under a nitrogen atmosphere. The basic ammoniumthioglycolate solution serves to solubilize the keratin and reduce thedisulfide cross-links. Cysteine thiol groups and cysteine thioglycolategroups are formed from the broken disulfide bonds. The nitrogenatmosphere serves to prevent oxidation and reformation of disulfidebonds. Heating is preferably followed by comminuting the hair particleswith a tissue homogenizer followed by further heating under a nitrogenatmosphere. A fine keratin suspension results.

The fine keratin suspension is centrifuged, and the supernatantcontaining a more soluble keratin fraction is collected and precipitatedout with acid. The precipitate is resuspended in ammonium hydroxide. Thekeratin solution is then cast as a thin film on a surface and allowed toair dry. The air serves to remove water, concentrate the keratin, andoxidize the cysteine thiol groups, forming disulfide bridges andstrengthening the film. Further crosslinking can be achieved usingchemical means such as glutaraldehyde. The resulting film is tough andinsoluble.

In one method, cut, washed, rinsed, and dried vertebrate hair isprovided. The hair is reduced with a reducing agent, such that some ofthe disulfide linkages are broken, and a more soluble keratin fractionand a less soluble keratin fraction formed. The more soluble keratinfraction is separated, collected, and concentrated, and the more solublekeratin fraction is deposited into a mold. The concentrated keratinsolution in the mold is exposed to an oxidizing or crosslinking agentand preferably dried. The free thiol groups are oxidized by theoxidizing agent or crosslinked by the crosslinking agent, and thekeratin strengthened by newly formed disulfide bonds. In another method,the concentrated keratin solution is either atomized into a cold mold ormixed with a polar solvent, emulsified, and freeze-dried to form anopen-cell material. The keratin solution is exposed to an oxidizing orcrosslinking agent, which cross-links and strengthens the material. Aporous keratin material remains.

In another method according to the present invention, the more solublekeratin solution is further concentrated, for example, by air drying orheating under sub-ambient pressure. The concentrated solution is pouredinto a mold and allowed to air dry. The air serves to remove water,concentrate the keratin, and oxidize the cysteine-thiol groups, formingdisulfide bridges and strengthening the keratin material. The resultingbulk keratin material is tough and insoluble. In another method, aliquid oxidizing agent such as hydrogen peroxide is used. In yet anothermethod, a crosslinking agent such as glutaraldehyde is used.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Keratin Preprocessing

In one method according to the present invention, hair is provided,preferably washed and unbleached. The hair can be harvested from a humanor animal source. The patient or a human donor is a preferred source ofhair for some medical applications, as hair from these sources is mostlikely to result in a non-antigenic product, although animal hair may beacceptable for certain individuals that do not have animal productallergy problems. In one method, the hair is washed with Versa-Clean™(Fisher Scientific, Pittsburgh, Pa.), rinsed with deionized water, andallowed to air dry.

Partial Oxidation of Keratin

The hair can be oxidized in peracetic acid or another suitable reagentsuch as H₂ O₂. A preferable treatment utilizes from 1% to 32% peraceticacid, at a temperature between about 0° C. and 100° C. for between 0.5and 24 hours. One method treats 30 grams of hair with 500 mL of 32%peracetic acid at 4° C. for 24 hours. This treatment with peracetic acidpartially oxidizes the naturally occurring disulfide linkages to producea protein with cysteic acid (--CH₂ SO₃ H) residues and remainingdisulfide linkages. The hair is recovered, preferably by filtrationthrough a coarse fritted glass filter, and rinsed numerous times withdeionized water until the rinse solution has a pH of 6.0 or higher. Thehair can then be dried in a vacuum oven at between 20° C. and 50° C. forbetween 0.5 and 5 days. One method dries the hair in a vacuum oven at40° C. for several days. The dried hair can then be pulverized andground into a fine powder. One method of grinding the hair uses aceramic mortar and pestle.

Reduction of Partially Oxidized Keratin

The keratin powder can be suspended in ammonium thioglycolate. In onemethod, pulverized keratin powder, derived from hair as described above,is suspended in about 3N ammonium hydroxide containing ammoniumthioglycolate. About 6 grams of keratin powder can be added per 75 mL ofammonium hydroxide. The strength of ammonium hydroxide is preferablyabout 3N, and the preferred concentration of ammonium thioglycolate isabout 11 mL (as thioglycolic acid) per 75 mL of ammonium hydroxide. Thesuspension can then be heated for a time sufficient to solubilize thesoluble fraction of the hair. The suspension, in one method, is heatedbetween 50° C. and 90° C. for between 1 and 24 hours, followed bycooling. According to another method, the suspension is heated to about60° C. for about 4 hours and cooled to room temperature. This treatmentcleaves the remaining disulfide linkages to produce cysteine residues inthe protein structure. At this point, the keratin protein contains bothcysteic acid residues and cysteine residues. The ratio of cysteic acidresidues and cysteine residues can be controlled by varying the time,temperature, and concentration of oxidant in the peracetic acidtreatment step previously described. The presence of sulfonic acidresidues imparts a hydrophilic property to the hair, as well as thefinal sheet product.

Separation of Partially Oxidized Reduced Keratin

After the oxidation/reduction treatment described above, a resistantkeratin fraction remains, consisting primarily of beta keratin. Thiskeratin fraction is preferably at least 80% beta keratin, mostpreferably greater than about 90% beta keratin. This fraction isinsoluble in the suspension and is removed in one method bycentrifugation at about 10,000 g for about 10 minutes. A thick,jelly-like supernatant remains and is discarded or, more preferably,kept for another use. The remaining insoluble fraction is composedmostly of the original cuticle (outer layer of hair shaft) and iscomposed primarily of beta keratin.

Acidification of Partially Oxidized, Reduced Keratin

The insoluble material is transferred to another container and acidifiedto a low pH. The pH is preferably less than about 3 and most preferablyless than about 1. In one method the pH is less than about 1 and theacid used can be either concentrated sulfuric acid or formic acid. Thistreatment disrupts hydrogen bonding of the cuticle structure of the hairshaft. The low pH disrupts the hydrogen bonds responsible for tightlybinding the keratin protein, resulting in its resistance to chemicalmodification. Applicants believe the acid at least partially unfolds orswells the protein, enhancing the solubility. The slurry preferably hasa concentration in the range of 0.001 grams/mL to 0.6 grams/mL. Theslurry most preferably has a concentration in the range of 0.2 grams/mLto 0.3 grams/mL.

Neutralization, Concentration and Oxidation of Partially Oxidized,Reduced Keratin

The unfolded or swelled keratin slurry can then be made slightly basicwith ammonium hydroxide, preferably about 6N strength. The slurry canthen be cast onto a flat surface and air dried to produce thecross-linked sheet. A preferred relative humidity range for drying isbetween 0% and 90%. The relative humidity is most preferably betweenabout 40% and 60% relative humidity. The partially unfolded, swelled,and partially solubilized keratin refolds upon addition of the baseduring drying, causing hydrogen bonding of the keratin. The free thiolgroups form disulfide linkages.

The insoluble keratin fraction from hair is thus treated so as to haveboth sulfonic acid groups and thiol groups, and is separated from thesoluble fraction. The insoluble fraction is treated with acid topartially unfold, swell, and solubilize the keratin, followed bytreatment with base and casting onto a flat surface to refold theprotein and form some disulfide bonds.

In an alternate method, in the acidification step, the keratin issuspended in a volatile acid, such as formic acid, having sufficientlylow pH to partially unfold or swell the keratin protein. In this method,the treatment with volatile base can be dispensed with. Theacidification step can be immediately followed by forming the keratinslurry into a sheet.

The resulting sheet may be cleansed of soluble reagents by repeatedtreatment with hot (boiling), deionized water, yielding a cross-linked,pure keratin sheet. The moist keratin sheet, formed of keratin derivedprimarily from beta keratin, has the consistency of moist paper. Thesheet produced will dry to a brittle material which can be rehydrated toa supple skin-like material, suitable for use as a sheet wound dressing.The sheet retains water and the rehydrated sheet has the look and feelof skin. In a preferred method of use, the sheet is hydratedsufficiently to allow the sheet to be draped over a wound.

Keratin Slurry Including Partially Oxidized Alpha and Beta KeratinFractions

In an alternate embodiment of the present invention, the keratincentrifugation step used to separate the soluble and insoluble partiallyoxidized keratin fractions is omitted and both fractions are used infurther processing. In one embodiment, both fractions are furtherprocessed together with acid as described above. In this method, bothsoluble and insoluble fractions are transferred to another container andacidified to a low pH. The unfolded or swelled keratin slurry can thenbe made slightly basic with ammonium hydroxide, preferably about 6Nstrength. The slurry can then be cast onto a flat surface and air driedto produce a cross-linked sheet. As an alternate method, in theacidification step, the keratin is suspended in a volatile acid, such asformic acid, as described previously. In this method, the treatment withvolatile base can be dispensed with. In one method, the thick slurryhaving both keratin fractions can be cast into a thin film as previouslydiscussed. The resulting product has a somewhat smoother texture than apure beta keratin derived product. In another method, the thick slurrycan be further concentrated and used to form a bulk keratin product asdiscussed below.

Use of Partially Oxidized Alpha Keratin Fraction

In one embodiment, the keratin in the keratin solution is at least 90%keratin derived from alpha keratin. The resulting keratin solutioncontaining partially oxidized keratin derived primarily from alphakeratin can be utilized as described above, in the formation of filmsand sheets. The alpha keratin found in hair is primarily crystallineprior to processing, but is primarily amorphous after processing andcross-linking. Thus the terms "alpha" and "beta" refer to the keratinprotein structures at the source, not necessarily the keratin proteinstructures after processing and cross-linking. The alpha keratin isderived primarily from hair cortex keratin while the beta keratin isderived primarily from hair cuticle keratin. A preferred method utilizeshair cortex keratin.

In another embodiment, the soluble keratin fraction is used to form asheet or film. After centrifugation such as described above, theinsoluble fraction can be set aside for other use. A thick, jelly-likesupernatant remains, which includes a soluble, partially oxidizedkeratin fraction derived primarily from alpha keratin. The keratinfraction is termed "soluble" as it is soluble in a basic, aqueoussolution. In a preferred method, "soluble" keratin refers to a keratinfraction soluble at a pH of 10 or greater, but which may be soluble atlower, basic pH. In a preferred method, "insoluble" keratin refers tokeratin insoluble at a pH of 10. The supernatant is collected. Thesupernatant can be treated with concentrated HCl until a gummyprecipitate is produced. The precipitate can be collected, washed withdeionized water, and dissolved in 15 mL of 3N ammonium hydroxide,forming a keratin solution.

Keratin Sheet Applications

Applicants believe the keratin product made according to this method issuitable for use as a cell-growth scaffold that is mitogenic and as anutrient support for cell growth. Applicants also believe thecross-linked keratin sheet can be used as a scaffold material for avariety of cells, including skin component cells (keratinocytes,fibroblasts, endothelial cells), osteoblasts, chondrocytes, andhepatocytes. In particular, applicants have shown that skin componentcells will grow and proliferate favorably on the keratin sheet.Applicants further believe the keratin sheet can be used as a diffusionmembrane and to encapsulate cells for various applications.

Anti-bacterial additives, ointments, and biologicals such as growthfactors or collagen can be added to the keratin sheet. Bactericidalointment or a suspension of antibiotics or biologicals can beimpregnated into the sheet dressing by passing a blade having theadditive at its front over the sheet, thereby evenly distributing theadditive over the sheet. Alternatively, the sheet material can be soakedin a solution containing the desired additive and the additive allowedto precipitate onto the surface of the sheet. The solvent can then beflashed off, leaving the sheet material impregnated and coated with thedesired additive.

Keratin Reduction without Previous Partial Oxidation Step

Clean, keratin-containing hair prepared as previously described can besuspended in a reducing agent. A preferred reducing agent is ammoniumthioglycolate. Other reducing agents believed suitable for use in thepresent invention include mercaptoethanol, dithiothreitol, thioglycerol,thiolactic acid, glutathione, cysteine, and sodium sulfide. In onemethod, the washed and cut hair, as described above, is suspended inabout 3N ammonium hydroxide containing ammonium thioglycolate. Theammonium hydroxide is believed to deprotonate the carboxylic acids andthe cysteine thiol groups, forming a polyanionic polymer havingincreased solubility in water. The ammonium hydroxide is believed topartially swell the keratin protein, exposing additional disulfidelinkages to reaction with thioglycolic acid. About 6 grams of hair canbe added per 75 mL of ammonium hydroxide. The strength of ammoniumhydroxide is preferably about 3N and the preferred concentration ofammonium thioglycolate is about 11 mL (as thioglycolic acid) per 75 mLof ammonium hydroxide. The suspension can then be heated for a timesufficient to solubilize the soluble fraction of the hair. Thesuspension in one method is heated between 50° C. and 90° C. for between1 and 24 hours, followed by cooling. In a preferred method, thesuspension is heated to about 60° C. for about 2 hours under a nitrogenatmosphere and homogenized with a tissue homogenizer, as will bedescribed in detail in the next section, for about 30 minutes until afine dispersion is produced.

Homogenizing/Comminuting of Reduced Keratin

Homogenizing, as the term is used herein, refers to the hair particlesbeing comminuted, that is, broken down into smaller particles using arotor/stator combination homogenizer blade. The reduced hair washomogenized in situ in the ammonium thioglycolate solution usingprocedures described in U.S. Pat. No. 5,358,935 (without using liquidN₂) incorporated by reference above. Hair is protected by a tough outerkeratin layer resistant to chemical treatment. The outer layer is formedof primarily beta keratin material. The homogenizing separates theouter, protective cuticle material from the inner, cortex material andcomminutes the hair to make small keratin particles. The cortex containskeratins moderately soluble in water, but keratins not normally exposedto water, lying within the protective cuticle. The cortex containsprimarily alpha keratin. The homogenization also exposes disulfide bondsto reactants such as thioglycolate. The dispersion in one method isfurther heated an additional two hours at 60° C. under a nitrogenatmosphere before being cooled to room temperature. The continuedheating step provides time for the ammonium thioglycolate to break andreduce the newly exposed cysteine disulfide linkages. A thick slurry isthe expected result in a preferred method. The heating speeds up thereduction of disulfide bonds. The nitrogen atmosphere prevents theoxidation of thiol groups by atmospheric oxygen. Applicants believe thistreatment cleaves disulfide linkages to produce cysteine andcysteine-thioglycolate disulfide residues in the protein structure.

Separation

After the treatment described above, a keratin fraction resistant to thetreatment remains, consisting primarily of beta keratin. This fractionis insoluble in the suspension and is removed in one method bycentrifugation at about 10,000 g for about 10 minutes. The insolublefraction can be set aside for other use. A supernatant remains, whichincludes a soluble keratin fraction derived primarily from alphakeratin. The keratin fraction is termed "soluble" as it is soluble in abasic, aqueous solution. In a preferred method, "soluble" keratin refersto a keratin fraction soluble at a pH of 10 or greater, but which may besoluble at lower, basic pH. In a preferred method, "insoluble" keratinrefers to keratin insoluble at a pH less than 10. The supernatant iscollected. The supernatant can be treated with concentrated HCl until agummy precipitate is produced. The precipitate can be collected, washedwith deionized water, and dissolved in 15 mL of 3N ammonium hydroxide,forming a keratin solution.

In one embodiment, the keratin in the keratin solution is at least 90%keratin derived from alpha keratin. The alpha keratin found in hair isprimarily crystalline prior to processing but is primarily amorphousafter processing and cross-linking. Thus the terms "alpha" and "beta"refer to the keratin protein structures at the source, not necessarilythe keratin protein structures after processing and cross-linking. Thealpha keratin is derived primarily from hair cortex keratin while thebeta keratin is derived primarily from hair cuticle keratin. Haircuticle keratin typically includes substantial color from the originalhair. Hair cortex keratin does not include the original hair color. Apreferred method utilizes hair cortex keratin.

Film and Sheet Formation

The solution can be cast into a thin film and allowed to air dry into across-linked film derived primarily from alpha keratin. The keratinre-forms disulfide bonds, giving the film added strength. Weaker bonds,such as hydrogen bonds, also impart strength to the keratin-based filmas the solution becomes more concentrated, bringing the keratin proteinsin closer proximity to one another. In one method, the solution ispoured onto a flat surface, for example a glass surface. In anothermethod, the solution is poured onto a rotating drum or moving belt.Pouring the solution onto a flat surface produces a thin, flat geometryresembling that of the final film. Forming the flat surface also createsa high surface to volume ratio, allowing air to penetrate into thesolution a substantial fraction of the total depth and volume.

Concentration and Oxidation

Air drying performs several functions. First, the air removes water,thereby concentrating the keratin solution. The more concentratedsolution increases the rate of formation and number of re-formeddisulfide bonds. The disulfide bonds formed or re-formed are notnecessarily between the same cysteine groups in the initial protein.Second, the air contains oxygen, which oxidizes the free thiol groups inthe protein, forming disulfide bonds. Other oxidizing gases can be usedin place of air, for example oxygen. Oxidizing liquids such as hydrogenperoxide are also suitable for oxidizing the free thiol groups. Third,the air allows the ammonium hydroxide to evaporate. The resultinglowered pH also helps reform the disulfide bonds. Fourth, the air allowssome excess thioglycolate to escape. When the film formation is carriedout in the presence of nitrogen rather than air, applicants believe thefilm formed has far fewer disulfide bonds, but that the film is boundwith hydrogen bonds, resulting in a film that is softer than the filmformed in the presence of oxygen. The resulting residual thiol activitywould provide sites for the incorporation of desirable thiol-containingbiological factors.

The concentration and oxidation causes the formation of a tough,insoluble material. Excess thioglycolate, and the disulfide ofthioglycolic acid, may remain in the film and can be removed throughextraction in boiling water. In one method of cleaning, the film isimmersed in boiling water for about 1.5 hours, changing the water every15 minutes. This cleaning is believed to remove mostly excess, unreactedthioglycolate as opposed to thioglycolate bound to the protein backbone.

Slow evaporation can also be used to remove ammonium hydroxide from thematerial, thereby lowering the pH and promoting cross-link formation.Reducing pH in itself causes increased cross-linking and precipitationof protein. An additional cross-linking agent such as glutaraldehyde canbe used to form cross-links other than disulfide cross-links. The use ofglutaraldehyde allows cross-linking without requiring the same degree ofconcentration or water removal as required for cross-linking relyingprimarily on disulfide bond formation and could also increase the finaldegree of crosslinking over the oxidation crosslinking procedure.

Another method for forming the disulfide cross-links includes the stepsof removing the water and ammonium hydroxide under vacuum. In onemethod, the soluble keratin fraction in solution is placed into achamber and a vacuum pulled on the chamber, removing much of the waterfrom the material. The water is volatilized at a low temperature,leaving behind a cross-linked keratin material

Uses

Applicants believe the resulting material can be formed into a thinfilm, wound dressing, or tissue-engineering scaffold. Another use is asa diffusion membrane, for example, for drug delivery. Yet another use isfor coating implantable devices, such as stents and maxillofacialimplants, with the non-antigenic cross-linked keratin film material. Theparts to be coated can be dipped in the keratinous solution, followed byair drying or other method to promote cross-linking. This gives strongadherence to the implant since cross-linking occurs on the actualimplant shape as a thin film. Yet another use is as an encapsulant toencapsulate cells. Individual cells can be encapsulated, allowing, forexample, the film to act as a nutrient supply, a mitogen, or a diffusionmembrane.

Further Concentration

In another method embodying the present invention, the resulting keratinsuspension is further concentrated. The resulting solution is preferablyconcentrated to a concentration of between about 0.1 and 0.5 grams permL, more preferably between about 0.3 and 0.4 grams per mL, and mostpreferably about 0.35 grams per mL. The concentrated keratin solutioncan be used to create a porous, open cell keratin scaffold, discussed indetail below, in the open cell section.

Keratin Slurry Including Alpha and Beta Keratin Fractions

In an alternate embodiment of the present invention, the keratincentrifugation step used to separate the soluble and insoluble keratinfractions is omitted, and both fractions are used in further processing.In one method, the thick slurry having both keratin fractions can becast into a thin film as previously discussed. The resulting product hasa somewhat rougher texture than the pure alpha keratin derived product.In another method, the thick slurry can be further concentrated and usedto form a bulk keratin product as previously discussed.

Keratin Slurry Including Alpha and Beta Keratin Fractions with FurtherAcid Treatment

In another embodiment of the present invention, the keratincentrifugation step used to separate the soluble and insoluble keratinfractions is omitted, and both fractions are further processed withacid. In this method, both soluble and insoluble fractions aretransferred to another container and acidified to a low pH. The pH ispreferably less than about 3 and most preferably less than about 1. Inone method, the pH is less than about 1, and the acid used can behydrochloric, concentrated sulfuric, or formic acid. Applicants believethe acid at least partially swells the protein, enhancing the solubilityof the insoluble fraction. The slurry preferably has a concentration inthe range of 0.001 grams/mL to 0.6 grams/mL. The slurry most preferablyhas a concentration in the range of 0.2 grams/mL to 0.3 grams/mL.

The unfolded or swelled keratin slurry can then be made slightly basicwith ammonium hydroxide, preferably about 6N strength. The slurry canthen be cast onto a flat surface and air dried to produce thecross-linked sheet. A preferred relative humidity range for drying isbetween 0% and 90%. The relative humidity is most preferably betweenabout 40% and 60% relative humidity. The partially unfolded or swelled,partially solubilized keratin refolds upon addition of the base duringdrying, causing hydrogen bonding of the keratin. The free thiol groupsform disulfide linkages. In an alternate method, glutaraldehyde can beadded to the partially solublized keratin to provide an increased degreeof crosslinking. As an alternate method, in the acidification step, thekeratin is suspended in a volatile acid, such as hydrochloric or formicacid, having sufficiently low pH to partially swell the keratin protein.In this method, the treatment with volatile base can be dispensed with.The acidification step can be immediately followed by forming thekeratin slurry into a sheet. The keratin slurry can also be furtherconcentrated for production of bulk keratin.

Keratin Slurry Including Primarily Beta Keratin with Further AcidTreatment

In another embodiment of the present invention, the keratincentrifugation step used to separate the soluble and insoluble keratinfractions is performed and the beta fraction is further processed withacid. In this method, the insoluble fraction is transferred to anothercontainer and acidified to a low pH. The pH is preferably less thanabout 3 and most preferably less than about 1. In one method, the pH isless than about 1 and the acid used can be hydrochloric, concentratedsulfuric, or formic acid. Applicants believe the acid at least partiallyswells the protein, enhancing the solubility of the insoluble fraction.The slurry preferably has a concentration in the range of 0.001 grams/mLto 0.6 grams/mL. The slurry most preferably has a concentration in therange of 0.2 grams/mL to 0.3 grams/mL.

The keratin slurry can then be made slightly basic with ammoniumhydroxide, preferably about 6N strength. The slurry can then be castonto a flat surface and air dried to produce the cross-linked sheet. Apreferred relative humidity range for drying is between 0% and 90%. Therelative humidity is most preferably between about 40% and 60% relativehumidity. The partially unfolded, swelled, partially solubilized keratinrefolds upon addition of the base during drying, causing hydrogenbonding of the keratin. The free thiol groups form disulfide linkages.In an alternate embodiment, glutaraldehyde can be added to the partiallysolublized keratin to provide an increased degree of crosslinking. As analternate method, in the acidification step, the keratin is suspended ina volatile acid, such as formic acid, having sufficiently low pH topartially swell the keratin protein. In this method, the treatment withvolatile base can be dispensed with. The acidification step can beimmediately followed by forming the keratin slurry into a sheet. Thekeratin slurry can also be further concentrated for production of bulkkeratin.

Keratin Open Cell and Bulk Materials

The present invention also includes methods for forming keratin bulkmaterials and porous open cell materials. The bulk material is suitablefor use as a cross-linked implantable device, which can be used formaxillofacial restoration, for example, for soft and hard tissuereplacement. The bulk material can also be used for orthopedicapplications such as bone filler and cartilage regeneration. A tubularform of the implanted material can also be used for neurologicalapplications such as nerve regeneration guides. The porous keratinmaterial can be used as a tissue-engineering scaffold.

The invention includes processes for forming solid and porous bulkkeratin materials. A keratinous material, such as human hair, isprovided. The hair is suspended in liquid and reduced with a reducingagent, breaking the disulfide bonds. A keratinous slurry is thepreferred result. The slurry, which can be further processed andpurified, is preferably further concentrated and deposited into a moldto form a solid part in the shape of the mold. Alternatively, the slurrycan be used to process open cell, foam materials using a variety ofmethods described in the literature. One technique uses a spray of theatomized keratin solution on the surface of a cooled mold, therebybuilding up a foam structure as described by Lo et al. for PLLA foamfabrication (H. Lo, S. Kadiyala, S. E. Guggino, and K. W. Leong, "Poly(L-lactic acid) foams with cell seeding and controlled-releasecapacity," J. Biomed. Mater. Res., Vol. 30, pp. 475-484, 1996). A secondtechnique, also developed for PLA/PGA polymers, uses freeze dryingemulsions of polymer solutions to process open cell polymer structures(K. E. Healy, K. Whang, and C. H. Thomas, "Method of fabricatingemulsion freeze-dried scaffold bodies and resulting products," U.S. Pat.No. 5,723,508, issued Mar. 3, 1998). A similar process can be modifiedusing the appropriate solvents and conditions to make an open cellkeratin scaffold. For example, the keratin is dissolved in a volatilenon-polar solvent and mixed with a volatile polar solvent in which thekeratin is insoluble. These two solvents are immiscible. An emulsion isgenerated using ultrasound or a homogenizer, frozen, and freeze-dried toremove the solvents. An oxidizing agent, such as air or a peroxide or acrosslinking agent such as glutaraldehyde, can be supplied to thekeratin material in the emulsion stage. The keratin concentration andoxidizing agent act to promote keratin cross-linking. The resultingkeratin cross-linked product is hard and porous, with a microstructuredependent on the exact method used.

Numerous characteristics and advantages of the invention covered by thisdocument have been set forth in the foregoing description. It will beunderstood, however, that this disclosure is, in many respects, onlyillustrative. Changes may be made in details, particularly in matters ofshape, size, choice of reagents, and ordering of steps without exceedingthe scope of the invention. The invention's scope is, of course, definedin the language in which the appended claims are expressed.

Experimental Results Partially Oxidized Keratin Product

In a first experiment, a sheet wound dressing not requiring a binder wasprepared from keratin derived from human hair. Human hair was obtainedfrom males aged 10 to 30 years, washed with Versa-Clean™ (FisherScientific, Pittsburgh, Pa.), rinsed with deionized water and allowed toair dry. This hair was subsequently chopped into approximately 0.25-inchto 2-inch lengths using shears. Thirty grams of this hair was treatedwith 500 mL of 32% peracetic acid (Aldrich Chemical, Milwaukee, Wis.) at4° C. for 24 hours. This treatment partially oxidized the disulfidelinkages. The hair was recovered by filtration through a coarse frittedglass filter and rinsed numerous times with deionized water until therinse solution was pH 6.0 or higher. The hair was dried under vacuum at40° C. for several days until completely dry and ground to a fine powderwith a ceramic mortar and pestle. The resulting material, 19 grams, wasfurther modified to produce a flexible, hydratable sheet composedprimarily of beta keratin.

Six grams of the pulverized, oxidized hair was suspended in 75 mL of 3Nammonium hydroxide containing 11 mL of ammonium thioglycolate (asthioglycolic acid). The suspension was heated to 60° C. for 4 hours andthen cooled to room temperature. This treatment cleaved the remainingdisulfide linkages to produce cysteine residues in the proteinstructure. An insoluble fraction remained, which was resistant tosolubilization by the ammonium hydroxide and ammonium thioglycolate. Theinsoluble fraction, comprised mostly of beta keratin, was isolated bycentrifugation at 10,000 g for 10 minutes. A thick, jelly-likesupernatant was removed from the centrifuged material and set aside.

The remaining insoluble fraction is composed mostly of the originalcuticle (outer layer of hair shaft) and is composed primarily of betakeratin. The insoluble material was transferred to a flask and acidifiedto a pH of between 0 and about 1 with concentrated sulfuric acid. Thepartially unfolded keratin was made slightly basic with 6N ammoniumhydroxide. The slurry was then cast onto a flat surface and air dried toproduce a cross-linked sheet. The resulting sheet was purified byimmersion in boiling water, which removed soluble reagents.

The use of keratin-containing materials in promoting wound healing wasdemonstrated in several experiments. In a first experiment, processedhuman hair was incubated with cell culture media. The media/hair mixturewas passed through a micro filter. Cell lines relevant to wound healing,including human microvascular endothelial cells, keratinocytes, andfibroblasts, were placed in cultures using this media extract.Significant proliferation of these wound healing cells was measured.Keratinocytes proliferated profusely, fibroblasts proliferated modestly,and endothelial cells proliferated profusely.

The mitogenic activity observed in fibroblast, keratinocyte, andendothelial cell cultures is additional evidence that the keratinousprotein material is not only biocompatible, but also mitogenic withthese cell lines. Additional biocompatibility was observed when keratinmicrofibrils were observed microscopically to be in direct contact withcells in the cell cultures. Specifically, keratinocytes and fibroblastswere observed to adhere to and congregate around microfibrils,indicating that desirous cell activity can be sustained on thisnaturally derived biopolymer matrix.

In a second experiment, processed human hair powder was incubated withcell culture media. The media/keratin mixture was passed through a microfilter. This media extract was used in proliferation studies withlymphocytes. The lymphocyte cell line did not proliferate, indicatingthe material to be non-immunogenic.

In a third experiment, processed human hair powder was incubated withcell culture media. The media/hair mixture was then passed through amicro filter. This media extract was used in proliferation studies withactivated T-lymphocytes. The T-lymphocytes proliferated normally,indicating no inhibition of the normal cell mediated immune response bythe keratin. This demonstrated no inhibition of this very importantfunction of immune cells.

In a fourth experiment, twenty-eight hairless rats were wounded oneither side of the dorsal midline with a dermatome, creating a partialthickness wound 0.12 inches in depth, and 2.0×4.0 cm in surface area.Half the wounds were treated with keratin powder, half were not, andboth halves were covered with polyurethane dressing. The wounds wereobserved for healing and biopsied at days 0, 2, 4 and 6 forhistochemical analysis. Planimetry studies showed 97% epithelializationof the keratin-treated wounds and 78% epithelialization of thenon-treated wounds at day 4. Histological analysis by H & E stainrevealed total epithelialization microscopically of the keratin-treatedwounds at day 2 and only partial epithelialization of the non-treatedwounds at day 2. Histological analyses at days 4 and 6 also revealed anacceleration of the epithelialization maturation process in thekeratin-treated wounds.

Human clinical studies are currently being performed on donor sites forskin grafts. One half of the donor wound site is treated with sterilizedkeratin powder and the opposite half treated in a standard fashion, withAdaptic™ non-adhering dressing from Johnson & Johnson. Preliminaryresults show the keratin-treated halves epithelialize sooner and maturemore rapidly. This was confirmed through both clinical observations andhistological results of four-millimeter punch biopsies. Subjectively,patients also have much less pain in the keratin-treated wounds.

Experimental Results, Keratin Product Without Partial Oxidation

In a fifth experiment, human hair was obtained from males aged 10 to 30years, washed with Versa-Clean™ (Fischer Scientific, Pittsburgh, Pa.),rinsed with deionized water and allowed to air dry. This hair wassubsequently chopped into approximately 0.25" to 2" lengths usingshears. Six grams of hair was suspended in 75 mL of 3N ammoniumhydroxide containing 11 mL of ammonium thioglycolate. This treatmentcleaved the disulfide cystine linkages to produce cysteine residues inthe protein structure. The suspension was heated to 60° C. for 2 hoursunder a nitrogen atmosphere and then homogenized with a tissuehomogenizer for 30 minutes until a fine dispersion was produced. Thedispersion was heated an additional 2 hours at 60° C. under a nitrogenatmosphere and then cooled to room temperature. The thick slurry wastransferred to a tube and centrifuged at 5000G for 10 minutes. Thesupernatant was treated with concentrated hydrochloric acid until agummy precipitate was produced. The precipitate was collected, washedwith deionized water and then dissolved in 15 mL of 3N ammoniumhydroxide.

This solution was then cast into a thin film and allowed to air dry. Thesolution was also further concentrated through evaporation, and castinto a solid block of material. The removal of the volatile base andwater from the solution and the action of the air upon the free thiolfraction of the soluble polypeptide caused the material to crosslinkinto an insoluble, tough material. The material was then purified andfreed of any remaining thioglycolic acid by extraction in boiling waterfor 1.5 hours.

In a sixth experiment, human hair was chemically treated as previouslydescribed. This produced a keratin solution that was then cast into asheet and oxidatively cross-linked to produce a non-soluble sheet ofkeratin. The sheet was purified by extraction with boiling water for 1.5hours, changing the water every 15 minutes. Segments of the sheetingwere then incubated with keratinocytes, fibroblasts, and humanmicrovascular endothelial cells. These cells were shown to grow andproliferate favorably on the keratin sheet. This indicates that skincomponent cells proliferate favorably in the presence of keratinsheeting produced by the above-described method.

Numerous characteristics and advantages of the invention covered by thisdocument have been set forth in the foregoing description. It will beunderstood, however, that this disclosure is, in many respects, onlyillustrative. Changes may be made in details, particularly in matters ofshape, size and ordering of steps without exceeding the scope of theinvention. The invention's scope is, of course, defined in the languagein which the appended claims are expressed.

What is claimed is:
 1. A keratin material comprising a porous, open cellkeratin structure comprising a cross-linked oxidized keratin, whereinsaid oxidized keratin contains cysteic acid residues.
 2. A keratinmaterial as recited in claim 1, wherein said structure includes akeratin structure having substantially spherical voids therein.
 3. Akeratin material as recited in claim 1, wherein said structure includesa plurality of packed, touching keratin spheres, said spheres beingsubstantially cross-linked together where touching and having voidstherebetween.
 4. A tissue-engineering scaffold comprising cross-linkedkeratin, wherein said cross-links comprise reformed disulfide bondsformed by oxidation of thiols resulting from breaking disulfide bonds.5. A tissue engineering scaffold as recited in claim 4, wherein saidcross-linked keratin contains cysteic acid residues.
 6. A bulk keratinproduct comprising:a keratin material and having a predefinedthree-dimensional geometry, wherein said keratin material comprises across-linked oxidized keratin that contains cysteic acid residues.
 7. Anopen cell scaffold comprising a keratin material cross-linked withdisulfide bonds, wherein said scaffold contains substantially sphericalvoids therein.
 8. An open cell scaffold comprising a keratin materialcross-linked with disulfide bonds, wherein said scaffold includes aplurality of packed, touching keratin spheres, said spheres beingsubstantially cross-linked together where touching and voidstherebetween.